Volume 106, number 1
ACTIVATION
FEBS LETTERS
October 1979
OF THE REACTION II COMPONENT OF P5 15 IN CHLOROPLASTS
BY PIGMENT SYSTEM 1
A. H. C. M. SCHAPENDONK+
and W. .I. VREDENBERG+
Laboratory for Plant Physiological Research, Agricultural University, Ceneraal Foulkesweg
The Netherlands
72, 6 703 BW Wageningen,
Received 9 July 1979
1. Introduction
The kinetics of the P.51.5 absorbance changes,
induced by single turnover saturating light flashes,
sofar have been interpreted to be a reflection of the
rise and decay of a tram-membrane
potential associated with the charge separation in photosystem 1
and photosystem 2 [l-3]. The multiphasic decay
kinetics of the PS 15 response after energisation with
multiple flashes, fired at high frequency, have been
suggested to be caused by an altered H+ permeability
at the moment at which the trans-membrane potential exceeds a critical value [4-81. Recently, however,
it has been shown that the multiphasic rise and decay
kinetics of the P51.5 response in intact chloroplasts,
which show close correspondence with the P5 15
response in algae [9], are the composite result of at
least two different processes: reaction I and reaction
II [lo]. The kinetics of reaction I, characterised by a
rise time of CO.5 ms and a decay rate constant of
9-13 s-l, have been shown to be similar to the
kinetics of the trans-membrane potential as measured
by means of microcapillary glass electrodes [lo-121.
Reaction II, characterised by a rise time of loo-150
ms and a decay rate constant of 1.6-2.5 s-r, has been
suggested to be a reflection of slow intra-membranal
Abbreviations:
AA, absorbance change(s); chl, chloropWl;
DCPIP, (2,6-dichlorophenol indophenol); DCMU, (3-(3,4dichlorophenyl)-l,l-dimethylurea);
PMS, (phenazine metho-
sulphate)
+ Present address: Department of Physical Plant Physiology,
Agricultural University, 6703 BW Wageningen,
The Netherlands
ElsevierfNorth-Holland
Biomedical Press
changes induced by field-dependent charge displacements in the vicinity of the P5 15 pigment complex.
It has been reported by some authors that photosystem 1 and photosystem 2 participate to a similar
extent in the generation of the Msrs [4,13]. Other
workers have attributed the &lsrs mainly to photosystem 1 activity [ 14,151. A slow phase of the flashinduced A5r5 increase in the O-100 ms time range
after a flash, was first observed in CWorella and was
shown to be mainly activated by system 1 [ 161.
Recently, a similar A increase has been observed in
intact chloroplasts [ 10,17-191. This communication
deals with results and analyses of the P5 15 L4 in
chloroplasts, under conditions at which one of the
photosystems was activated. The slow component
of the P5 15 aA (reaction II) is suggested to be
induced by (changes in) a local electric field associated
with the charge interaction between the acceptor of
photosystem 1 and cytochrome for plastocyanine.
2. Materials and methods
Intact chloroplasts were isolated from fresh grown
spinach leaves by the method in [lo]. The assay
medium contained 330 mM sorbitol, 2 mM Hepes
(pH 7.6) 0.5 mM K2HP04, 10 mM NaHCOa and
10 mM MgCl*. Broken chloroplasts were obtained by
osmotic shocking in water, containing 10 mM KCl.
The assay medium contained 330 mM sorbitol,
30 mM tricine (pH 7.7) and 17.5 mM KCl. Chloroplast
concentration was equivalent to -50 pg chl.ml-‘.
Chloroplasts were kept in the dark for 1 h. Pre-incubation and measurements occurred at 2°C. aA~,-,--540,
257
Volume 106, number 1
FEBS LETTERS
induced either by single flashes or series of 3 successive
flashes with a half-life time of 8 gs and at wavelengths
>665 nm or at 717 nm, were measured as in [lo].
Inhibition of the electron transport between photosystem 2 and photosystem 1 was achieved by preincubation of the sample in the presence of KCN,
by the method in [20], or by addition of DCMU.
3. Results
Figure la and 1b show representative examples of
the aA sls in intact chloroplasts upon saturating single
turnover light flashes, which excite both photo~stems
((a) excitation light of wavelengths X65 nm), or preferentially photosystem 1 ((b) light of narrow wavelength band -7 17 nm), respectively. According to
analyses, illustrated and discussed in [IO], the 5 15 nm
response can be resolved into two different components. These have been interpreted to be due to rise
and decay of the trans-membrane electric field (reaction I) and the rise and decay of a local electric field
in the membrane core (reaction II), respectively. The
result of these analyses with respect to the response
shown in fig. 1a,b are given by the solid curve (reaction I) and broken curve (reaction II). The response
of reaction I, characterised by a single exponential
decay, in this case was obtained by measuring the ALI
in flashes fired at a repetition rate of 4 s-l after 3
pre-~lum~ating flashes which were necessary to
a
b
Fig-l. Time courses of the AA,,, in intact chloropl~s upon
gumption
with single turnover light flashes at wavelengths
>665 nm (a) and at a narrow region -717 nm (b). Flash
frequency was 0.05 Hz (noisy curves) or 3 Hz (solid curves).
In case of the latter 3 pre-illuminating flashes were flued (see
text). The dotted lines represent the resultant kinetic pattern
(reaction II) after subtraction of the solid curves (reaction I)
from the overall response.
258
October 1979
complete and saturate reaction II [IO]. The response
of reaction II sub~quently was obtained by subtracting the one of reaction I from the overall response.
Figure 1 shows that the magnitude of reaction I and
II in 717 nm light (system 1) is -50% and 75%, respectively, of the magnitude in light absorbed by both
photosystems.
Figure 2a,b show the d4 upon illumination with
3 successive light flashes of wavelengths >665 nm and
of 7 17 nm, respectively, in intact chloroplasts. The
time intervals between the flashes was 100 ms. The
dotted curves represent the P515 absorbance responses
upon a single flash. As the absorbance decay in the
dark, 300 ms after a flash, is exclusively due to that
of reaction I1 (cf. fig. l), it appears, according to fig.2a
and conclusive with results obtained with double flash
experiments [IO], that reaction II is saturated by a
single light flash of wavelengths >665 nm. This apparently is not the case when iight of 717 nm wavelength
is used. Repetitive excitation of photosystem 1 gives
rise to a further increase of reaction II in the second
and third flash (fig.2b). It should be mentioned that,
with 717 nm light, the third and the second flash were
not completely saturating. Therefore it might be that
the saturation level of reaction II can be reached after
two successive excitations with system I light.
Figure 3 shows the responses in broken chloroplasts,
in the absence (a) and presence of electron transport
inhibitors DCMU (b,c) or KCN (d,e). In the presence
of DCMU (b), the extent of the P5 1.5response is
-50% of the M associated with reaction I in the
absence of DCMU (a). The M in the presence of
DCMU unfortunately cannot be analysed into components with sufficient accuracy. In general, reaction
I can only be measured accurately by fast repetitive
flashes [IO]. However, as would be expected, no signal
is observed under this condition in the presence of
DCMU (see also [ 131). Addition of DCPIPjascorbate
creates a condition in which a total recovery of the
P515 response is measured (tig.3c). A similar response
has been measured after addition of PMS to DCMUpoisoned chloroplasts (not shown). The response
measured after pre-incubation of the chbroplasts in
the presence of 30 mM KCN is shown in fig.3d. The
magnitude of the signal is -40% of that of the reaction I response in the control experiment. The decay
of the response in the presence of KCN appears to be
faster than the decay of the signal in EMU-poisoned
Volume 106, number I
October 1979
FEBS LFTTERS
a
a
C
-a
b
J
f
h
e
-‘r’----
Fig.3. Time courses of the bA5,5 upon illumination with
single turnover light flashes at wavelengths >665 nm fled at
a frequency of 0.05 Hz in broken chlorophrsts. ChIoroplasts
were suspended in 330 mM sorbitol, 30 mM tricme (pH 7.6)
and either 17.5 mM KC1 (a-c) or 17.5 mM KCN (d,e). (a)
Control experiment; (b) in the presence of 2 /.LMDCMU after
pre-illumination with 4 flashes; (c) in the presence of 2 MM
DCMU, 3.3 mM ascorbate and 48 PM DCPIP; (d) after preincubation in 30 mM KCN; (e) after pre-incubation in KCN
and subsequent addition of 3.3 mM ascorbate and 48 PM
DCPIP.
Fig.2. Time courses of the AA,,, in intact chloroplasts upon
illumination with triple flashes at wavelengths >665 nm (a)
and at a narrow wavelength region -717 nm (b). Time span
between separate flashes was 100 ms. Series of 3 flashes were
fired at a frequency of 0.05 Hz. The dotted lines represent
the responses of single flashes fired at 0.05 Hz.
chloroplasts and suggests the complete absence of
reaction II and a somewhat faster decay rate of reaction I. Addition of DCPIP/ascorbate (fig.3e), in this
case, creates a condition in which the flash-induced
aA equals the reaction I response of the control,
except for the somewhat faster decay. The dark kinetics suggest the absence of reaction II in KCN-inhibited
chloroplasts in the presence of DCPIP/ascorbate.
259
Volume 106, number 1
FEBS LETTERS
AA
Fig.4. Resolution of the AA 515in intact chloroplasts upon
illumination with single turnover light flashes at wavelengths
>665 nm in the absence (a) and presence (b) of 200 nM
valinomycin, into two components: reaction I and reaction
II. Flash frequency was 0.05 Hz (overall responses) or 3 Hz
(curves I). In case of the latter 3 pre-illuminating flashes
were fired. The dotted lines represent the resultant kinetic
patterns (reaction II) after subtraction of curves I from the
overall responses.
Figure 4 shows the resolved responses of the reaction I and II component of the Msrs in the absence
and presence of 200 nM valinomycin.
4. Discussion
The results presented in this work indicate that
the reaction I and reaction II components of the P.515
aA upon single turnover saturating light flashes in
dark-adapted chloroplasts are differently activated
by photosystem 1 and 2. According to the data of
fig. 1,2 it appears that the magnitude of the fast rise
of reaction I in system 1 (717 nm) light is -50% of
the magnitude measured in light absorbed by both
photosystems. Moreover the magnitude of the PS 15
response measured in DCMU- or KCN-treated chloroplasts (fig.3b and d, respectively) upon flash light
absorbed by both systems is -50% of the reaction I
response in the absence of the inhibitors (fig.3a).
Illumination of DCMU-poisoned broken chloroplasts
with 7 17 nm light flashes under these conditions did
not result in an absorbance response in the absence
of electron donors for system I. We therefore conclude, in agreement with the interpretation
[ 131, that
the response measured in the presence of these electron-transport inhibitors is brought about by charge
separation in pigment system 2.
According to fig.3a,c,e, reaction I can be restored
260
October 1979
completely in DCMU- or KCN-poisoned chloroplasts
by the addition of electron donors for system I. Thus
it seems reasonable to conclude that reaction I is
equally activated by photosystem 1 and photosystem
2. This conclusion is consistent with the suggestion
that reaction I is the electrochromic response of the
PS 15 complex upon generation and decay of the
trans-membrane electric potential [lo]. It has been
shown that this potential, measured with micro-electrodes is equally activated by both pigment systems
[111.
Reaction II appears to depend quite differently on
system 1 and system 2 illumination. According to the
data of fig. 1,2, the magnitude of reaction II in a single
flash of system 1 light is 75% and 65%, respectively,
of the (saturated) magnitude of reaction II in light
absorbed by both systems. We found for a variety of
preparations that excitation of system 1 causes a
60-80% activation of reaction II. Figure 2 shows in
addition that saturation of reaction II can be achieved
after double or triple excitation of system 1. This
would suggest that the trans-membrane electric field,
which in case of system 1 excitation is only 50% of
the field generated by both systems (see before),
modifies the membrane conformation necessary for
a maximal reaction II response. Thus we conclude
that reaction II is mainly dependent on a process
activated by system 1 excitation. This conclusion is
substantiated by the observation (fig.3) that reaction
II is relatively small, or even absent in DCMU- (fig.2b)
and KCN-treated (fig.2c) chloroplasts, respectively,
and can be completely restored in the presence of
DCMU after addition of electron donors for system
1. The decay of the P5 15 response in the presence of
DCMU (fig.2b) suggests a contribution of reaction II,
which in this case is -20% of the maximal response
under conditions at which electron transfer through
system 1 is possible (fig.2a,d). Whether this small contribution of reaction II has to be attributed to system 1
activity cannot be said with certainty. Although system 1 excitation under these conditions was found
not to result in a P5 15 response, it is possible that, upon
excitation of both systems, the reduced acceptor of
system 2 is oxidized in the dark by a component
which in its reduced form serves as a (weak) donor
for oxidized carriers at the donor side of system 1.
Thus, upon excitation, some electron transport
through system 1 would be possible with the conse-
Volume 106, number 1
FEBS LETTERS
appearance
of a small reaction II (and reaction
I). The results in [ 191 indeed suggest that electron
transfer in system 1 is partially restored in DCMUtreated chloroplasts in the presence of dithionite.
Our results do not permit definite conclusions
about the process which is responsible for the appearance of reaction II. A slow A sr5 increase has been
reported to occur in pre-illuminated chloroplasts [ 181,
which has been suggested to be caused by a proton
translocation at the oxidizing site of the plastoquinone
pool. In our experiments however the resolved slow
A,4, associated with reaction II, occurs under conditions at which plastoquinone is in the oxidized form
(system 1 illumination).
It has been suggested that the slow decay component of P5 15, which according to our analyses is
attributed to that of reaction II, represents the decay
of the transmembrane potential generated by cyclic
electron transport in system 1 [ 191. However, this
suggestion is difficult to reconcile with the fact (fig.4)
that the decay of reaction II is unaltered whereas its
magnitude is suppressed in the presence of low concentrations of valinomycin. Additional evidence that
the slow decay component of P5 15 (reaction II) is difficult to interpret in terms of a trans-membrane potential has been given in [lo]. We have presumed that
changes in the mutual orientation of fixed charges due
to conformational
changes in the membranal core,
affect the interaction of the associated electric fields
with the P.5 15 pigment complex. The absence of
reaction II in KCN-poisoned chloroplasts, in the
presence of electron donors for system 1 (fig.3e)
would suggest a functional role of plastocyanine or
cytochrome fin this process. Thus it seems likely that
changes in charge densities occurring at these sites
during electron transport from system 2 or from artificial donors (in the absence of KCN). cause alterations in strength and orientation of inner-membrane
electric fields. This interpretation
is in agreement
with the observation [21] that a distinct component
of the PS 15 response is dependent on the redox condition of a component with a midpoint potential of
+385 mV [21], which is about equal to the midpoint
potential of cytochrome f and plastocyanin.
quent
October 1979
Acknowledgements
The investigation was supported in part by the
Foundation for Biophysics (Stichting voor Biofysica),
financed by the Netherlands Organization for the
Advancement of Pure Research (ZWO).
References
Ill Witt, H. T. (1971) Quart. Rev. Biophys. 4, 365-477.
121 Junge, W. and Witt, H. T. (1968) Z. Naturforsch. 23b,
244-254.
131 Boeck, M. and Witt, H. T. (1972) in: Proc. IInd Int.
Congr. Photosynthesis (Forti, G. et al. eds) vol. 2,
pp. 903-911, W. Junk, The Hague.
[41 Schliephake, W., Junge, W. and Witt, H. T. (1968)
Z. Naturforsch. 23b, 1571-1578.
[51 Rumberg, B. and Siggel, U. (1968) Z. Naturforsch. 23b,
239-244.
[61 Junge, W., Rumberg, B. and Schroder, H. (1970)
Eur. J. Biochem. 14,575-581.
[71 Schmid, R., Schavit, R. and Junge, W. (1976) Biochim.
Biophys. Acta 430, 145-153.
[81 Girault, G. and Galmiche, J. (1978) Biochim. Biophys.
Acta 502,430-444.
191 Joliot, P. and Delosme, R. (1974) Biochim. Biophys.
Acta 357, 267-284.
[lOI Schapendonk, A. H. C. M., Vredenberg, W. J. and Tonk,
W. J. M. (1979) FEBS Lett. 100,325-330.
[Ill Vredenberg, W. J. and Tonk, W. J. M. (1974) FEBS Lett.
42,236-239.
[121 Bulychev, A. A. and Vredenberg, W. J. (1975) Biochim.
Biophys. Acta 423, 548-556.
1131 Renger, G. and Wolff, C. (1975) Z. Naturforsch. 3Oc,
161-171.
[I41 Satoh, K. and Katoh, S. (1977) Plant Cell Physiol. 18,
1077-1087.
[I51 Kulandaivelu, G. and Senger, H. (1976) Biochim.
Biophys. Acta 430, 94-104.
1161 Joliot, P. and Delosme, R. (1974) Biochim. Biophys.
Acta 357, 267-284.
[I71 Horvath, G., Droppa, M., Mustardy, L. A. and
Faludi-Daniel, A. (1978) Planta 141, 239-244.
[I81 Velthuys, B. R. (1978) Proc. Natl. Acad. Sci. USA 75,
6031-6035.
[W Crowther, D., Mills, J. D. and Hind, G. (1979)
FEBS Lett. 98, 386-390.
WV Quitrakul, R. and Izawa, S. (1973) Biochim. Biophys.
Acta 305, 105-118.
1211 Malkin, R. (1978) FEBS Lett. 87,329-333.
261